The long-term goal of this Phase II proposal is the production of new ketolide antibiotics with potent antibacterial activity against macrolide-susceptible and macrolide-resistant bacterial pathogens of humans. In Phase I research, we successfully developed a biological process for production of 15-R-6-deoxyerythronolide B, the biochemical precursor of 15-R-erythromycins (R=various chemical groups), that involves expression of the 6-deoxyerythronolide B (DEBS) polyketide synthase (PKS) genes in an Escherichia coli strain carrying the requisite PKS substrate supply genes. In Phase II this process will be optimized for large-scale production of the desired 15-R-6-deoxyerythronolide B (15-R-6dEB) by feeding the 5R-3-hydroxy-2-methylpentanoic acid N-acetylcysteamine thioester ("diketide-SNAC") to an E. coli strain expressing the engineered DEBS1 module 2/DEBS2/DEBS3 genes. The resulting 15-R-6dEB will be used subsequently to produce a lead ketolide Kosan has discovered in partnership with another company. The specific aims for the Phase II research are: 1) to determine the relationship between the titer of polyketide produced and the level of DEBS PKS, substrate supply enzymes and substrates. These data will help us design and construct a recombinant E. coli strain that produces >100 mg/L of 15-R-6dEB in a diketide-fed, shake flask fermentation. 2) To isolate, by random mutagenesis of an E. coli strain bearing DEBS PKS and substrate supply genes, mutant strains with a >10-fold increase in 15-R-6dEB titer in a diketide-fed, shake flask fermentation. The improved genetic background of these mutants is expected to enhance the performance of the optimum arrangement of the DEBS PKS and substrate supply genes created in Specific Aim 1. 3) To introduce the optimal metabolically engineered DEBS PKS and substrate supply genes from Specific Aim 1 into the E. coli strain from Specific Aim 2 to create an E. coli recombinant strain that produces >250 mg/L of 15-R-6dEB in a diketide-fed, shake flask fermentation. 4) To optimize the physiological and process parameters for maximum production of 15-R-6dEB at >1 g/L by high cell density E. coli cultures in a 2 liter stirred fermentor. This will be done with a strain obtained through achievement of Specific Aim 3. Several of the ketolide series of analogs based on 15-R-erythromycin A have excellent in vitro and in vivo antibacterial activity, comparable to or better than the leading ketolides in current clinical trials or approved by the FDA for specific uses. We intend to move the best compound to pre-clinical testing in collaboration with our partner. [unreadable] [unreadable]